Nuclear envelope fractions prepared from isolated Novikoff ascites tumor nuclei have 10 to 12 bands of peptides between 43,000 and 68,000 MW which constitute most of the protein of the sample. In particular, a peptide of 43,000 MW (Band 43) comprises a significant portion of the nuclear envelope peptide subunits. Although mitochondrial content of the isolated nuclei represents 0.6 percent or less of the original mitochondrial enzyme content of the cell homogenate, some mitochondrial protein may co-purify with the nuclear envelope fraction. Electrophoretic separation of mitochondrial, microsomal, and nuclear envelope polypeptides on slab polyacrylamide gel sheets in an SDS-tris-glycine discontinuous buffer system demonstrated that Band 43 is not a measurable component of mitochondria and, if present in microsomes, is not a major component. Relationship of Band 43 to chromatin was investigated by tracing its separation into the soluble phase when isolated nuclei were extracted with detergents, salts and acids. Those procedures which are known to partially or totally remove nuclear envelope material from nuclei also solubilized Band 43. Thus, by indirect proof, Band 43 is apparently derived from envelope material, is basic in nature, and can be solubilized by non-ionic detergents.